Genotyping‐by‐sequencing approaches to characterize crop genomes: choosing the right tool for the right application

In the last decade, the revolution in sequencing technologies has deeply impacted crop genotyping practice. New methods allowing rapid, high‐throughput genotyping of entire crop populations have proliferated and opened the door to wider use of molecular tools in plant breeding. These new genotyping‐by‐sequencing (GBS) methods include over a dozen reduced‐representation sequencing (RRS) approaches and at least four whole‐genome resequencing (WGR) approaches. The diversity of methods available, each often producing different types of data at different cost, can make selection of the best‐suited method seem a daunting task. We review the most common genotyping methods used today and compare their suitability for linkage mapping, genomewide association studies (GWAS), marker‐assisted and genomic selection and genome assembly and improvement in crops with various genome sizes and complexity. Furthermore, we give an outline of bioinformatics tools for analysis of genotyping data. WGR is well suited to genotyping biparental cross populations with complex, small‐ to moderate‐sized genomes and provides the lowest cost per marker data point. RRS approaches differ in their suitability for various tasks, but demonstrate similar costs per marker data point. These approaches are generally better suited for de novo applications and more cost‐effective when genotyping populations with large genomes or high heterozygosity. We expect that although RRS approaches will remain the most cost‐effective for some time, WGR will become more widespread for crop genotyping as sequencing costs continue to decrease.     

BioNano genome mapping of individual chromosomes supports physical mapping and sequence assembly in complex plant genomes

The assembly of a reference genome sequence of bread wheat is challenging due to its specific features such as the genome size of 17 Gbp, polyploid nature and prevalence of repetitive sequences. BAC‐by‐BAC sequencing based on chromosomal physical maps, adopted by the International Wheat Genome Sequencing Consortium as the key strategy, reduces problems caused by the genome complexity and polyploidy, but the repeat content still hampers the sequence assembly. Availability of a high‐resolution genomic map to guide sequence scaffolding and validate physical map and sequence assemblies would be highly beneficial to obtaining an accurate and complete genome sequence. Here, we chose the short arm of chromosome 7D (7DS) as a model to demonstrate for the first time that it is possible to couple chromosome flow sorting with genome mapping in nanochannel arrays and create a de novo genome map of a wheat chromosome. We constructed a high‐resolution chromosome map composed of 371 contigs with an N50 of 1.3 Mb. Long DNA molecules achieved by our approach facilitated chromosome‐scale analysis of repetitive sequences and revealed a ~800‐kb array of tandem repeats intractable to current DNA sequencing technologies. Anchoring 7DS sequence assemblies obtained by clone‐by‐clone sequencing to the 7DS genome map provided a valuable tool to improve the BAC‐contig physical map and validate sequence assembly on a chromosome‐arm scale. Our results indicate that creating genome maps for the whole wheat genome in a chromosome‐by‐chromosome manner is feasible and that they will be an affordable tool to support the production of improved pseudomolecules.     

Design of efficient simplified genomic DNA and bisulfite sequencing in large plant populations

The next generation sequencing enables generation of high resolution and high throughput data for structure sequence of any genome at a fast declining cost. This opens opportunity for population based genetic and genomic analyses. In many applications, whole genome sequencing or re-sequencing is unnecessary or prohibited by budget limits. The Reduced Representation Genome Sequencing (RRGS), which sequences only a small proportion of the genome of interest, has been proposed to deal with the situations. Several forms of RRGS are proposed and implemented in the literature. When applied to plant or crop species, the current RRGS protocols shared a key drawback that a significantly high proportion (up to 60%) of sequence reads to be generated may be of non-genomic origin but attributed to chloroplast DNA or rRNA genes, leaving an exceptional low efficiency of the sequencing experiment. We recommended and discussed here the design of optimized simplified genomic DNA and bisulfite sequencing strategies, which may greatly improves efficiency of the sequencing experiments by bringing down the presentation of the undesirable sequencing reads to less than 10% in the whole sequence reads. The optimized RAD-seq and RRBS-seq methods are potentially useful for sequence variant screening and genotyping in large plant/crop populations.     

Sequence-based linkage analysis

The rapid decrease in the cost of DNA sequencing will enable its use for novel applications. Here, we investigate the use of DNA sequencing for simultaneous discovery and genotyping of polymorphisms in family linkage studies. In the proposed approach, short contiguous segments of genomic DNA, regularly spaced across the genome, are resequenced in each pedigree member, and all sequence polymorphisms discovered within a pedigree are used as genetic markers. We use computer simulations consistent with observed human sequence diversity to show that segments of 500-1,000 base pairs, spaced at intervals of 1-2 Mb across the genome, provide linkage information that equals or exceeds that of traditional marker-based approaches. We validate these results experimentally by implementing the sequence-based linkage approach for chromosome 19 in CEPH pedigrees.     

Family‐assisted inference of the genetic architecture of major histocompatibility complex variation

With their direct link to individual fitness, genes of the major histocompatibility complex (MHC) are a popular system to study the evolution of adaptive genetic diversity. However, owing to the highly dynamic evolution of the MHC region, the isolation, characterization and genotyping of MHC genes remain a major challenge. While high‐throughput sequencing technologies now provide unprecedented resolution of the high allelic diversity observed at the MHC, in many species, it remains unclear (i) how alleles are distributed among MHC loci, (ii) whether MHC loci are linked or segregate independently and (iii) how much copy number variation (CNV) can be observed for MHC genes in natural populations. Here, we show that the study of allele segregation patterns within families can provide significant insights in this context. We sequenced two MHC class I (MHC‐I) loci in 1267 European barn owls (Tyto alba), including 590 offspring from 130 families using Illumina MiSeq technology. Coupled with a high per‐individual sequencing coverage (~3000×), the study of allele segregation patterns within families provided information on three aspects of the architecture of MHC‐I variation in barn owls: (i) extensive sharing of alleles among loci, (ii) strong linkage of MHC‐I loci indicating tandem architecture and (iii) the presence of CNV in the barn owl MHC‐I. We conclude that the additional information that can be gained from high‐coverage amplicon sequencing by investigating allele segregation patterns in families not only helps improving the accuracy of MHC genotyping, but also contributes towards enhanced analyses in the context of MHC evolutionary ecology.     

Development of genomic resources in support of sequencing, assembly, and annotation of the catfish genome

Major progress has been made in catfish genomics including construction of high-density genetic linkage maps, BAC-based physical maps, and integration of genetic linkage and physical maps. Large numbers of ESTs have been generated from both channel catfish and blue catfish. Microarray platforms have been developed for the analysis of genome expression. Genome repeat structures are studied, laying grounds for whole genome sequencing. USDA recently approved funding of the whole genome sequencing project of catfish using the next generation sequencing technologies. Generation of the whole genome sequence is a historical landmark of catfish research as it opens the real first step of the long march toward genetic enhancement. The research community needs to be focused on aquaculture performance and production traits, take advantage of the unprecedented genome information and technology, and make real progress toward genetic improvements of aquaculture brood stocks.     

Novel Genomic Tools and Modern Genetic and Breeding Approaches for Crop Improvement

In recent past, genomic tools especially molecular markers have been extensively used for understanding genome dynamics as well for applied aspects in crop breeding. Several new genomics technologies such as next generation sequencing (NGS), high-throughput marker genotyping, -omics technologies have emerged as powerful tools for understanding genome variation in crop species at DNA, RNA as well as protein level. These technologies promise to provide an insight into the way gene(s) are expressed and regulated in cell and to unveil metabolic pathways involved in trait(s) of interest for breeders not only in model-/major- but even for under-resourced crop species which were once considered “orphan” crops. In parallel, genetic variation for a species present not only in cultivated genepool but even in landraces and wild species can be harnessed by using new genetic approaches such as advanced-backcross QTL (AB-QTL) analysis, introgression libraries (ILs), multi-parent advanced generation intercross (MAGIC) population and association genetics. The gene(s) or genomic regions, responsible for trait(s) of interest, identified either through conventional linkage mapping or above mentioned approaches can be introgressed or pyramided to develop superior genotypes through molecular breeding approaches such as marker-assisted back crossing (MABC), marker assisted recurrent selection (MARS) and genome wide selection (GWS). This article provides an overview on some recent genomic tools and novel genetic and breeding approaches as mentioned above with a final aim of crop improvement.     

Goat domestication and breeding: a jigsaw of historical,biological and molecular data with missing pieces

Domestic goats (Capra hircus) are spread across the five continents with a census of 1 billion individuals. The worldwide population of goats descends from a limited number of bezoars (Capra aegagrus) domesticated 10 000 YBP (years before the present) in the Fertile Crescent. The extraordinary adaptability and hardiness of goats favoured their rapid spread over the Old World, reaching the Iberian Peninsula and Southern Africa 7000 YBP and 2000 YBP respectively. Molecular studies have revealed one major mitochondrial haplogroup A and five less frequent haplogroups B, C, D, F and G. Moreover, the analysis of autosomal and Y‐chromosome markers has evidenced an appreciable geographic differentiation. The implementation of new molecular technologies, such as whole‐genome sequencing and genome‐wide genotyping, allows for the exploration of caprine diversity at an unprecedented scale, thus providing new insights into the evolutionary history of goats. In spite of a number of pitfalls, the characterization of the functional elements of the goat genome is expected to play a key role in understanding the genetic determination of economically relevant traits. Genomic selection and genome editing also hold great potential, particularly for improving traits that cannot be modified easily by traditional selection.     

Multiplex restriction amplicon sequencing: a novel next‐generation sequencing‐based marker platform for high‐throughput genotyping

To enable rapid selection of traits in marker‐assisted breeding, markers must be technically simple, low‐cost, high‐throughput and randomly distributed in a genome. We developed such a technology, designated as Multiplex Restriction Amplicon Sequencing (MRASeq), which reduces genome complexity by polymerase chain reaction (PCR) amplification of amplicons flanked by restriction sites. The first PCR primers contain restriction site sequences at 3’‐ends, preceded by 6‐10 bases of specific or degenerate nucleotide sequences and then by a unique M13‐tail sequence which serves as a binding site for a second PCR that adds sequencing primers and barcodes to allow sample multiplexing for sequencing. The sequences of restriction sites and adjacent nucleotides can be altered to suit different species. Physical mapping of MRASeq SNPs from a biparental population of allohexaploid wheat (Triticum aestivum L.) showed a random distribution of SNPs across the genome. MRASeq generated thousands of SNPs from a wheat biparental population and natural populations of wheat and barley (Hordeum vulgare L.). This novel, next‐generation sequencing‐based genotyping platform can be used for linkage mapping to screen quantitative trait loci (QTL), background selection in breeding and many other genetics and breeding applications of various species.     

Development,validation and genetic analysis of a large soybean SNP genotyping array

Cultivated soybean (Glycine max) suffers from a narrow germplasm relative to other crop species, probably because of under‐use of wild soybean (Glycine soja) as a breeding resource. Use of a single nucleotide polymorphism (SNP) genotyping array is a promising method for dissecting cultivated and wild germplasms to identify important adaptive genes through high‐density genetic mapping and genome‐wide association studies. Here we describe a large soybean SNP array for use in diversity analyses, linkage mapping and genome‐wide association analyses. More than four million high‐quality SNPs identified from high‐depth genome re‐sequencing of 16 soybean accessions and low‐depth genome re‐sequencing of 31 soybean accessions were used to select 180 961 SNPs for creation of the Axiom^® SoyaSNP array. Validation analysis for a set of 222 diverse soybean lines showed that 170 223 markers were of good quality for genotyping. Phylogenetic and allele frequency analyses of the validation set data indicated that accessions showing an intermediate morphology between cultivated and wild soybeans collected in Korea were natural hybrids. More than 90 unanchored scaffolds in the current soybean reference sequence were assigned to chromosomes using this array. Finally, dense average spacing and preferential distribution of the SNPs in gene‐rich chromosomal regions suggest that this array may be suitable for genome‐wide association studies of soybean germplasm. Taken together, these results suggest that use of this array may be a powerful method for soybean genetic analyses relating to many aspects of soybean breeding.     

From deep sequencing to viral tagging: Recent advances in viral metagenomics

Culture‐independent high‐throughput sequencing has provided unprecedented insights into microbial ecology, particularly for Earth's most ubiquitous and diverse inhabitants – the viruses. A plethora of methods now exist for amplifying the vanishingly small amounts of nucleic acids in natural viral communities in order to sequence them, and sequencing depth is now so great that viral genomes can be detected and assembled even amid large concentrations of non‐viral DNA. Complementing these advances in amplification and sequencing is the ability to physically link fluorescently labeled viruses to their host cells via high‐throughput flow sorting. Sequencing of such isolated virus–host pairs facilitates cultivation‐independent exploration of the natural host range of viruses. Within the next decade, as these technologies become widespread, we can expect to see a systematic expansion of our knowledge of viruses and their hosts.     

高通量测序技术在罕见病分子诊疗中的 应用及临床实例分析

罕见病病种繁多,且表型复杂多样,不仅仅体现在疾病间的不同,同一种疾病的不同患者在表型上也可能大相径庭。这种普遍存 在的遗传异质性和临床异质性,使罕见病的诊疗极具挑战。近年来,在后人类基因组计划时代,各种测序技术快速发展,使得大规模测 序如疾病目标基因集测序、全外显子组测序、全基因组测序等成为了现实。高通量测序技术可实现对多个靶基因进行高通量平行测序, 有效节约了成本与时间,越来越广泛地应用到临床疾病分子诊疗领域。分析传统测序技术与高通量测序技术的优缺点,介绍罕见病诊疗 中常用的高通量测序策略,并结合临床实例,综述高通量测序技术在罕见病诊疗中的应用。     

Genomic Characterization of DArT Markers Based on High-Density Linkage Analysis and Physical Mapping to the Eucalyptus Genome

Diversity Arrays Technology (DArT) provides a robust, high throughput, cost-effective method to query thousands of sequence polymorphisms in a single assay. Despite the extensive use of this genotyping platform for numerous plant species, little is known regarding the sequence attributes and genome-wide distribution of DArT markers. We investigated the genomic properties of the 7,680 DArT marker probes of a Eucalyptus array, by sequencing them, constructing a high density linkage map and carrying out detailed physical mapping analyses to the Eucalyptus grandis reference genome. A consensus linkage map with 2,274 DArT markers anchored to 210 microsatellites and a framework map, with improved support for ordering, displayed extensive collinearity with the genome sequence. Only 1.4 Mbp of the 75 Mbp of still unplaced scaffold sequence was captured by 45 linkage mapped but physically unaligned markers to the 11 main Eucalyptus pseudochromosomes, providing compelling evidence for the quality and completeness of the current Eucalyptus genome assembly. A highly significant correspondence was found between the locations of DArT markers and predicted gene models, while most of the 89 DArT probes unaligned to the genome correspond to sequences likely absent in E. grandis, consistent with the pan-genomic feature of this multi-Eucalyptus species DArT array. These comprehensive linkage-to-physical mapping analyses provide novel data regarding the genomic attributes of DArT markers in plant genomes in general and for Eucalyptus in particular. DArT markers preferentially target the gene space and display a largely homogeneous distribution across the genome, thereby providing superb coverage for mapping and genome-wide applications in breeding and diversity studies. Data reported on these ubiquitous properties of DArT markers will be particularly valuable to researchers working on less-studied crop species who already count on DArT genotyping arrays but for which no reference genome is yet available to allow such detailed characterization.     

Analysis of ancient human genomes

High‐capacity sequencing technologies have dramatically reduced both the cost and time required to generate complete human genome sequences. Besides expanding our knowledge about existing diversity, the nature of these technologies makes it possible to extend knowledge in yet another dimension: time. Recently, the complete genome sequence of a 4,000‐year‐old human from the Saqqaq culture of Greenland was determined to 20‐fold coverage. These data make it possible to investigate the population affinities of this enigmatic culture and, by identifying several phenotypic traits of this individual, provide a limited glimpse into how these people may have looked. While undoubtedly a milestone in ancient DNA research, the cost to generate an ancient genome, even from such an exceptionally preserved specimen, remains out of reach for most. Nonetheless, recently developed DNA capture methods, already applied to Neanderthal and fossil human mitochondrial DNA, may soon make large‐scale genome‐wide analysis of ancient human diversity a reality, providing a fresh look at human population history.     

Sequencing technologies and genome sequencing

The high-throughput - next generation sequencing (HT-NGS) technologies are currently the hottest topic in the field of human and animals genomics researches, which can produce over 100 times more data compared to the most sophisticated capillary sequencers based on the Sanger method. With the ongoing developments of high throughput sequencing machines and advancement of modern bioinformatics tools at unprecedented pace, the target goal of sequencing individual genomes of living organism at a cost of $1,000 each is seemed to be realistically feasible in the near future. In the relatively short time frame since 2005, the HT-NGS technologies are revolutionizing the human and animal genome researches by analysis of chromatin immunoprecipitation coupled to DNA microarray (ChIP-chip) or sequencing (ChIP-seq), RNA sequencing (RNA-seq), whole genome genotyping, genome wide structural variation, de novo assembling and re-assembling of genome, mutation detection and carrier screening, detection of inherited disorders and complex human diseases, DNA library preparation, paired ends and genomic captures, sequencing of mitochondrial genome and personal genomics. In this review, we addressed the important features of HT-NGS like, first generation DNA sequencers, birth of HT-NGS, second generation HT-NGS platforms, third generation HT-NGS platforms: including single molecule Heliscope™, SMRT™ and RNAP sequencers, Nanopore, Archon Genomics X PRIZE foundation, comparison of second and third HT-NGS platforms, applications, advances and future perspectives of sequencing technologies on human and animal genome research.     

A draft genome sequence of the pulse crop chickpea (Cicer arietinum L.)

Cicer arietinum L. (chickpea) is the third most important food legume crop. We have generated the draft sequence of a desi‐type chickpea genome using next‐generation sequencing platforms, bacterial artificial chromosome end sequences and a genetic map. The 520‐Mb assembly covers 70% of the predicted 740‐Mb genome length, and more than 80% of the gene space. Genome analysis predicts the presence of 27 571 genes and 210 Mb as repeat elements. The gene expression analysis performed using 274 million RNA‐Seq reads identified several tissue‐specific and stress‐responsive genes. Although segmental duplicated blocks are observed, the chickpea genome does not exhibit any indication of recent whole‐genome duplication. Nucleotide diversity analysis provides an assessment of a narrow genetic base within the chickpea cultivars. We have developed a resource for genetic markers by comparing the genome sequences of one wild and three cultivated chickpea genotypes. The draft genome sequence is expected to facilitate genetic enhancement and breeding to develop improved chickpea varieties.     

quaddRAD: a new high‐multiplexing and PCR duplicate removal ddRAD protocol produces novel evolutionary insights in a nonradiating cichlid lineage

The identification of thousands of variants across the genomes and their accurate genotyping are crucial for estimating the genetic parameters needed to address a host of molecular ecological and evolutionary questions. With rapid advances of massively parallel high‐throughput sequencing technologies, several methods have recently been developed to access genomewide data on population variation. One of the most successful and widely used techniques relies on the combination of restriction enzymes and sequencing‐by‐synthesis: restriction‐site‐associated DNA sequencing (RADSeq). We developed a new, more time‐ and cost‐efficient double‐digest RAD paired‐end protocol (quaddRAD) that simplifies and speeds up the identification of PCR duplicates and permits large‐scale multiplexing. Assessing its performance on a technical data set, we also applied the quaddRAD method on population samples of a Neotropical cichlid fish lineage (Archocentrus centrarchus) to assess its genetic structure and demographic history. While we identified allopatric interlake genetic divergence, most likely driven by drift, no signature of sympatric divergence was detected. This differs from what has been observed in the clade of Midas cichlids (Amphilophus citrinellus spp.), another cichlid lineage that inhabits the same lakes and shares a similar demographic history, but has evolved into small‐scale adaptive radiations via sympatric speciation. We demonstrate that quaddRAD is a robust and efficient method for genotyping a massive number and widely overlapping set of loci with high accuracy. Furthermore, the results on A. centrarchus open new research avenues providing an ideal system to investigate genome‐level mechanisms that could alter the speciation potential of different but closely related cichlid lineages.     

A 3.9-centimorgan-resolution human single-nucleotide polymorphism linkage map and screening set

Recent advances in technologies for high-throughout single-nucleotide polymorphism (SNP)-based genotyping have improved efficiency and cost so that it is now becoming reasonable to consider the use of SNPs for genomewide linkage analysis. However, a suitable screening set of SNPs and a corresponding linkage map have yet to be described. The SNP maps described here fill this void and provide a resource for fast genome scanning for disease genes. We have evaluated 6,297 SNPs in a diversity panel composed of European Americans, African Americans, and Asians. The markers were assessed for assay robustness, suitable allele frequencies, and informativeness of multi-SNP clusters. Individuals from 56 Centre d'Etude du Polymorphisme Humain pedigrees, with >770 potentially informative meioses altogether, were genotyped with a subset of 2,988 SNPs, for map construction. Extensive genotyping-error analysis was performed, and the resulting SNP linkage map has an average map resolution of 3.9 cM, with map positions containing either a single SNP or several tightly linked SNPs. The order of markers on this map compares favorably with several other linkage and physical maps. We compared map distances between the SNP linkage map and the interpolated SNP linkage map constructed by the deCode Genetics group. We also evaluated cM/Mb distance ratios in females and males, along each chromosome, showing broadly defined regions of increased and decreased rates of recombination. Evaluations indicate that this SNP screening set is more informative than the Marshfield Clinic's commonly used microsatellite-based screening set.